Project Summary A primary focus of the NIH BRAIN Initiative is to develop new technologies for large scale high resolution imaging of brain activity. Imaging, as opposed to traditional electrode array based measurements, promise much greater spatial resolution and specificity. Excellent fluorescent calcium indicators have been developed to make it possible to image activity with high spatial and temporal resolution. But calcium signaling is secondary to electrical signaling, which is the primary form of information transfer within the brain circuitry. However, current tools for imaging electrical activity have severe limitations, primarily due to limited sensitivity and poor signal to noise. This proposal focusses on the development of new hybrid voltage sensors, hGEVIs, based on genetically encoded fluorescent proteins (FPs) and linked organic fluorescence quenchers. The system is designed so that the fluorophore is localized to the outer surface of the membrane while the tethered quencher translocates across the membrane in response to voltage. Based on our previous experience with an all organic voltage sensor composed of a tethered bichromophoric fluorophore quencher (TBFQ), we know that huge voltage sensitivities can be achieved. Our proposed hGEVIs promise to be bright and produce extraordinary voltage sensitivity. They will have the additional benefit of allowing the sensors to be targeted to specific brain circuits or regions. This new class of voltage indicators could make electrical imaging as widespread and accessible as calcium imaging.